Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons

Expressions of voltage-gated sodium channels Nav1.1 and Nav1.2, encoded by SCN1A and SCN2A genes, respectively, have been reported to be mutually exclusive in most brain regions. In juvenile and adult neocortex, Nav1.1 is predominantly expressed in inhibitory neurons while Nav1.2 is in excitatory neurons. Although a distinct subpopulation of layer V (L5) neocortical excitatory neurons were also reported to express Nav1.1, their nature has been uncharacterized. In hippocampus, Nav1.1 has been proposed to be expressed only in inhibitory neurons. By using newly generated transgenic mouse lines expressing Scn1a promoter-driven green fluorescent protein (GFP), here we confirm the mutually exclusive expressions of Nav1.1 and Nav1.2 and the absence of Nav1.1 in hippocampal excitatory neurons. We also show that Nav1.1 is expressed in inhibitory and a subpopulation of excitatory neurons not only in L5 but all layers of neocortex. By using neocortical excitatory projection neuron markers including FEZF2 for L5 pyramidal tract (PT) and TBR1 for layer VI (L6) cortico-thalamic (CT) projection neurons, we further show that most L5 PT neurons and a minor subpopulation of layer II/III (L2/3) cortico-cortical (CC) neurons express Nav1.1 while the majority of L6 CT, L5/6 cortico-striatal (CS), and L2/3 CC neurons express Nav1.2. These observations now contribute to the elucidation of pathological neural circuits for diseases such as epilepsies and neurodevelopmental disorders caused by SCN1A and SCN2A mutations.

SCN1A and SCN2A genes, respectively, have been reported to be mutually exclusive in most brain regions. In juvenile and adult neocortex, Nav1.1 is predominantly expressed in inhibitory neurons while Nav1.2 is in excitatory neurons. Although a distinct subpopulation of layer V (L5) neocortical excitatory neurons were also reported to express Nav1.1, their nature has been uncharacterized. In hippocampus, Nav1.1 has been proposed to be expressed only in inhibitory neurons. By using newly generated transgenic mouse lines expressing Scn1a promoter-driven green fluorescent protein (GFP), here we confirm the mutually exclusive expressions of Nav1.1 and Nav1.2 and the absence of Nav1.1 in hippocampal excitatory neurons. We also show that Nav1.1 is expressed in inhibitory and a subpopulation of excitatory neurons not only in L5 but all layers of neocortex. By using neocortical excitatory projection neuron markers including FEZF2 for L5 pyramidal tract (PT) and TBR1 for layer VI (L6) cortico-thalamic (CT) projection neurons, we further show that most L5 PT neurons and a minor subpopulation of layer II/III (L2/3) cortico-cortical (CC) neurons express Nav1.1 while the majority of L6 CT, L5/6 cortico-striatal (CS), and L2/3 CC neurons express Nav1.2. These observations now contribute to the elucidation of pathological neural circuits for diseases such as epilepsies and neurodevelopmental disorders caused by SCN1A and SCN2A mutations.

Introduction
Voltage-gated sodium channels (VGSCs) play crucial roles in the generation and propagation of action potentials, contributing to excitability and information processing (Catterall, 2012). They consist of one main pore-forming alpha-and one or two subsidiary beta-subunits that regulate kinetics or subcellular trafficking of the alpha subunits. Human has nine alpha (Nav1.1-Nav1.9) and four beta (beta-1-beta-4) subunits. Among alphas, Nav1.1, Nav1.2, Nav1.3, and Nav1.6, encoded by SCN1A, SCN2A, SCN3A, and SCN8A, respectively, are expressed in central nervous system. SCN3A is mainly expressed embryonically (Brysch et al., 1991), and SCN1A, SCN2A, and SCN8A are major alphas after birth. Although these three genes show mutations in a wide spectrum of neurological diseases such as epilepsy, autism spectrum disorder (ASD), and intellectual disability, two of those, SCN1A and SCN2A, are major ones (reviewed in Yamakawa, 2016;Meisler et al., 2021). To understand the circuit basis of these diseases, it is indispensable to know the detailed distributions of these molecules in the brain.
We previously reported that expressions of Nav1.1 and Nav1.2 seem to be mutually exclusive in many brain regions (Yamagata et al., 2017). In adult neocortex and hippocampus, Nav1.1 is dominantly expressed in medial ganglionic eminence-derived parvalbumin-positive (PV-IN) and somatostatin-positive (SST-IN) inhibitory neurons (Ogiwara et al., 2007;Lorincz and Nusser, 2008;Ogiwara et al., 2013;Li et al., 2014;Tai et al., 2014;Tian et al., 2014;Yamagata et al., 2017). In the neocortex, some amount of Nav1.1 is also expressed in a distinct subset of layer V (L5) excitatory neurons , but their natures were unknown. In the hippocampus, Nav1.1 seems to be expressed in inhibitory but not in excitatory neurons (Ogiwara et al., 2007;Ogiwara et al., 2013). In contrast, a major amount of Nav1.2 (~95%) is expressed in excitatory neurons including the most of neocortical and all of hippocampal ones, and a minor amount is expressed in caudal ganglionic eminence-derived inhibitory neurons such as vasoactive intestinal polypeptide (VIP)-positive ones (Lorincz and Nusser, 2010;Yamagata et al., 2017;Ogiwara et al., 2018). However, a recent study reported that a subpopulation (more than half) of VIP-positive inhibitory neurons is Nav1.1-positive (Goff and Goldberg, 2019).
VGSCs are mainly localized at axons and therefore it is not always easy to identify their origins, the soma. To overcome this, here in this study we generated bacterial artificial chromosome (BAC) transgenic mouse lines that express GFP under the control of Scn1a promoters, and we carefully Arrows indicate the start sites and orientation of transcription of Scn1a. (B) Western blot analysis for Scn1a-GFP and endogenous Nav1.1. The whole cytosolic fractions from 5W Scn1a-GFP brains (lines #184 and #233) were probed with anti-GFP and their membrane fractions were probed with anti-Nav1.1 antibodies. β-Tubulin was used as an internal control. pA, polyadenylation signal; Tg, hemizygous Scn1a-GFP transgenic mice; Wt, wild-type littermates.
The online version of this article includes the following source data and figure supplement(s) for figure 1: Source data 1. Raw and annotated immunoblots for Figure 1B. investigated the GFP/Nav1.1 distribution in mouse brain. Our analysis confirmed that expressions of Nav1.1 and Nav1.2 are mutually exclusive and that in neocortex Nav1.1 is expressed in both inhibitory and excitatory neurons while in hippocampus only in inhibitory but totally absent in excitatory neurons. Furthermore by using a transcription factor FEZF2 (FEZ family zinc finger protein 2 transcriptional factor), also referred to as Fezl, Fez1, Zfp312, and Fez, as a marker for L5 pyramidal tract (PT) neurons (Inoue et al., 2004;Chen et al., 2005;Chen et al., 2008;Molyneaux et al., 2005;Lodato et al., 2014;Matho et al., 2021), and a transcription factor TBR1 which suppresses FEZF2 expression and therefore does not overlap with FEZF2 (Han et al., 2011;McKenna et al., 2011;Matho et al., 2021), we found that most of L5 FEZF2-positive neurons are GFP-positive while L5/6 TBR1-positive neurons are largely GFP-negative and Nav1.2-positive. These results proposed that Nav1.1 is expressed in L5 PT while Nav1.2 in L5/6 non-PT neurons such as L5/6 cortico-striatal (CS) and L6 cortico-thalamic (CT) projection neurons. A majority of L2/3 excitatory neurons express Nav1.2 but a minor subpopulation are GFP-positive, suggesting that most of cortico-cortical (CC) projection neurons express Nav1.2 but the distinct minor population express Nav1.1. These results refine the expression loci of Nav1.1 and Nav1.2 in the brain and should contribute to the understanding of circuit mechanisms for diseases caused by SCN1A and SCN2A mutations.

Generation and verification of Scn1a-GFP transgenic mouse lines
Scn1a-GFP founder mice were generated from C57BL/6J zygotes microinjected with a modified Scn1a-GFP BAC construct harboring all, upstream and downstream, Scn1a promoters (Nakayama et al., 2010; Figure 1A) (see Materials and methods for details). Western blot analysis ( Figure 1B) and immunohistochemistry (Figure 1-figure supplement 1) showed robust GFP expression and mostly normal expression levels of Nav1.1 in Scn1a-GFP mouse lines #184 and #233. Both lines showed a similar distribution of chromogenic GFP immunosignals across the entire brain ( Figure 2A-H), and a similar distribution was also obtained in fluorescence detection of GFP ( Figure (Ogiwara et al., 2007;Tai et al., 2014) (see also Figure 8), were scattered in stratum oriens, pyramidale, radiatum, lucidum, and lacunosummoleculare of the CA (cornu ammonis) fields, hilus and molecular layer of dentate gyrus. Of note, somata of dentate granule cells were apparently GFP-negative. CA1-3 pyramidal cells were twined around with fibrous GFP immunosignals. We previously reported that the fibrous Nav1.1 signals clinging to somata of hippocampal CA1-3 pyramidal cells were disappeared by conditional elimination of Nav1.1 in PV-INs but not in excitatory neurons, and therefore concluded that these Nav1.1-immunopositive fibers are axon terminals of PV-INs . As such, GFP signals are fibrous but do not form cell shapes in the CA pyramidal cell layer ( Figure 2C, G, K and Figure 2-figure supplement 1C, G), and therefore these CA pyramidal cells themselves are assumed to be GFP-negative. These observations further confirmed our previous proposal that hippocampal excitatory neurons are negative for Nav1.1 (Ogiwara et al., 2007;Ogiwara et al., 2013). In cerebellum ( Figure 2D, H, L and Figure 2-figure supplement 1D, H), GFP signals appeared in Purkinje, basket, and deep cerebellar nuclei cells, again consistent to the previous reports (Ogiwara et al., 2007;Ogiwara et al., 2013). In the following analyses, we used the line #233 which shows stronger GFP signals than #184.
The online version of this article includes the following source data and figure supplement(s) for figure 2:

Nav1.1 is expressed in both excitatory and inhibitory neurons in neocortex but only in inhibitory neurons in hippocampus
In the neocortex of Scn1a-GFP mouse, a large number of cells with GFP-positive somata (GFP-positive cells) were broadly distributed across all cortical layers (Figure 3 and Figure 3-figure supplement 1). Intensities of GFP signals in primary somatosensory cortex (S1) at L2/3 are much higher than other areas such as primary motor cortex (M1) (Figure 3-figure supplement 1), however the cell population (density) of GFP-positive cells did not differ in these areas indicating that GFP signals for GFPpositive cells are stronger in S1 at L2/3. Although GFP signals are strong in PV-INs (see Figure 8), cell density of PV-INs is not specifically high at S1 area and therefore most cells with strong GFP signals in S1 at L2/3 may not be PV-INs but excitatory neurons.
In order to know the ratio of GFP-positive cells among all neurons, we further performed immunohistochemical staining using NeuN-antibody on Scn1a-GFP mouse at P15 and cells were counted at M1 and S1 (  (Weyer and Schilling, 2003). Therefore, NeuN-positive cells do not represent all neurons in neocortex as well. NeuN/GFP-double negative neurons could even exist and therefore above figure (Figure 3-figure supplement 2B) may deviate from the real ratios of GFP-positive cells among all neurons.
Double in situ hybridization of Scn1a and GFP mRNAs showed that these signals well overlap in both neocortex and hippocampus of Scn1a-GFP mice ( Figure 6), further supporting that the     GFP signals well represent endogenous Scn1a/Nav1.1 expression. Again, in neocortex Scn1a and GFP mRNAs seem to be expressed in a number of neurons including some of excitatory pyramidal cells, while in hippocampus they are absent in excitatory neurons such as CA1-3 pyramidal cells and dentate granule cells. All of these distributions of Scn1a and GFP mRNAs in Scn1a-GFP transgenic mouse brain are consistent to our previous report of regional distributions of Scn1a mRNA in wildtype mouse (Ogiwara et al., 2007).
To investigate the ratio of inhibitory neurons in GFP-positive cells, we generated and examined Scn1a-GFP and vesicular GABA transporter Slc32a1 (Vgat)-Cre  double transgenic mice in which Slc32a1-Cre is expressed in all GABAergic inhibitory neurons and visualized by floxed tdTomato transgene ( Figure 7). In the neocortex at 4W, 23% (L2/3), 28% (L5), and 27% (L6) of GFP-positive cells were Tomato-positive inhibitory neurons and 73% (L2/3), 77% (L5), and 83% (L6) of Tomato-positive cells were GFP-positive ( Figure 7C and Supplementary file 1i, j). These results suggest that a significant subpopulation of neocortical excitatory neurons also express Nav1.1. Our previous observation that Nav1.1 is expressed in callosal axons of neocortical excitatory neurons  supports that a subpopulation of L2/3 CC neurons express Nav1.1. Unlike in neocortex, in the hippocampus most of GFP-positive cells were Tomato-positive, 98% (CA1) and 94% (DG), and majorities of Tomato-positive GABAergic neurons are GFP-positive, 93% (CA1) and 77% (DG). These results further confirmed that in hippocampus Nav1.1 is expressed in inhibitory neurons but not in excitatory neurons. Although somata of pyramidal cells in CA2/3 region are weakly GFPpositive in this and some other experiments ( Figure 7B and  Figure 7C) among GFP-positive cells would be explained by us counting a cell as PV-positive if their PV immunosignals are moderate and a significant subpopulation of such cells are known to be excitatory neurons (Jinno and Kosaka, 2004;Tanahira et al., 2009;Matho et al., 2021). Quantitative analysis of GFP signal intensity and area size of cells revealed that GFP signal intensities in PV-positive cells were significantly higher than those in PV-negative cells and GFP signal intensities in SST-positive cells were lower than those in PV-positive cells but similar to PV/SST-double negative cells ( Figure 9 and Supplementary file 1n-p). These results indicate that Nav1.1 expression level in PV-INs is significantly higher than those in excitatory neurons and PV-negative GABAergic neurons including SST-INs.
Neocortical PT and a subpopulation of CC projection neurons express Nav1.1 Neocortical excitatory neurons can be divided into functionally distinct subpopulations, a majority of those are pyramidal cells which have axons of long-range projections such as L2/3 CC, L5 PT, L5/6 CS, and L6 CT projection neurons (Shepherd, 2013). Although PT neurons also project their axon collaterals to ipsilateral striatum, CS neurons project bilaterally to ipsi-and contralateral striata. A transcription factor FEZF2 is expressed in L5 PT neurons, forms their axonal projections, and defines their targets (Inoue et al., 2004;Chen et al., 2005;Chen et al., 2008;Lodato et al., 2014). Most of PT neurons are FEZF2-positive (Molyneaux et al., 2005;Matho et al., 2021). We previously reported that a subpopulation of neocortical L5 pyramidal neurons is Nav1.1-positive , but their natures were unclear. To investigate those, here we performed immunohistochemical staining of FEZF2 and GFP on Scn1a-GFP mouse brains (Figures 12 and 13, Supplementary file 1uv and w). In L5 where a major population of FEZF2-positive cells locate ( Figure 12A), a majority of FEZF2-positive neurons were GFP-positive (83% and 96% of FEZF2-positive neurons were GFP-positive at P15 and 4W, respectively) ( Figure 12B, middle panels and Supplementary file 1v). In L2/3, FEZF2-positive cells were scarce ( Figure 12A). In L6, a certain number of FEZF2-positive cells exist but overlaps of FEZF2 and GFP signals are much less compared to those in L5 (Figure 12 and Supplementary file 1u-w). Quantitative analyses revealed that FEZF2/GFP-double positive cells in L5 showed significantly GFP-positive somata in which AISs but not somata are positive for Nav1.1. All images are oriented from pial surface (top) to callosal (bottom). Scale bars: 100 μm (A), 50 μm (B, C). (D) Cell counting of three Scn1a-GFP mice. Bar graphs indicating the percentage of cells with GFP-and Nav1.1-positive/ negative somata and AISs per cells with ankyrinG-positive AISs (left panel), the percentage of cells with GFP-positive/negative somata per cells with ankyrinG-positive AISs and Nav1.1-positive somata and/or AISs (middle panel), and the percentage of cells with Nav1.1-positive/negative somata and/ or AISs per cells with ankyrinG-positive AISs and GFP-positive somata (right panel) in L2/3, L5, and L6 (see also Supplementary file 1b-d). Only cells with ankyrinG-positive AISs were counted. Nav1.1 immunosignals were occasionally observed in somata, but in such cases Nav1.1 signals were always observed in their AISs if visible by ankyrinG staining. Note that 99% (L2/3), 99% (L5), and 97% (L6) of cells with Nav1.1-positive AISs have GFP-positive somata (middle panel), but only half or less of cells with GFP-positive somata have Nav1.1-positive AISs (right panel). L2/3, L5: neocortical layer II/III and V. AnkG, ankyrinG; +, positive; −, negative.
The online version of this article includes the following source data and figure supplement(s) for figure 4: Source data 1. Numerical source data for Figure 4D.    Triple immunofluorescent staining of parasagittal sections from P15 Scn1a-GFP mouse brain (line #233) by mouse anti-GFP (green), rabbit anti-Nav1.1 (magenta), and goat anti-ankyrinG (cyan) antibodies. Regions at hippocampus were shown. Note that green fluorescent protein (GFP) and Nav1.1 immunosignals mostly overlap at somata. CA1, cornu ammonis 1; CA2, cornu ammonis 2; CA3, cornu ammonis 3; DG, dentate gyrus. Images are oriented from pial surface (top) to callosal lower GFP immunosignal intensities and larger signal areas (soma sizes) compared to those of FEZF2negative/GFP-positive cells (Figure 13 and Supplementary file 1x), indicating that L5 PT neurons showed lower Nav1.1 expression compared to other neurons such as PV-INs (see also Figure 9). Soma sizes of GFP-positive cells in L6 were overall smaller than those of FEZF2/GFP-double positive cells in L5, and there was no statistically significant difference in size between the FEZF2-positive/negative subpopulations. However, FEZF2/GFP-double positive cells still showed lower intensity of GFP signals compared to FEZF2-negative/GFP-positive cells (Figure 13 and Supplementary file 1x).
We further performed triple immunostaining of FEZF2, Nav1.1, and ankyrinG on Scn1a-GFP mice at P15, and found that 11% of FEZF2-positive cells have Nav1.1-positive AIS in L5 of Scn1a-GFP mouse neocortex (Figure 12-figure supplement 1 and Supplementary file 1y, z, aa). The low ratios of FEZF2/Nav1.1-double positive cells are most possibly due to immunohistochemically undetectable low levels of Nav1.1 expression in these excitatory neurons.
We also performed triple immunostaining of FEZF2, Nav1.2, and ankyrinG on Scn1a-GFP mice at P15 (Figure 12-figure supplement 2 and Supplementary file 1ab, ac, ad, ae). The staining showed that 20% of neurons (cells with ankyrinG-positive AISs) in L5 are FEZF2-positive and a half of L5 FEZF2positive cells have Nav1.2-positive AISs. Together with the observation that most of FEZF2-positive cells are GFP-positive (Figure 12), these results indicate that a subpopulation of FEZF2-positive PT neurons may express both Nav1.1 and Nav1.2.
We additionally performed triple immunostaining of FEZF2, GFP, and Nav1.2 on Scn1a-GFP mice at P15 (Figure 12-figure supplement 3 and Supplementary file 1af), showing that in L5 74% of FEZF2positive cells are GFP-positive but a majority of their AISs are Nav1.2-negative. The ratios of Nav1.2positive cells among FEZF2-positive cells obtained in the triple immunostaining of FEZF2, GFP, and Nav1.2 ( Figure 12-figure supplement 3) are 27% (L5) and 40% (L6). These results further support the above notion that a subpopulation of FEZF2-positive PT neurons may express both Nav1.1 and Nav1.2. Although further studies such retrograde tracking analyses are required to confirm and figure out the detailed circuits, all these results propose that the majority of L5 PT neurons express Nav1.1.
The majority of CT, CS, and CC projection neurons express Nav1.2 TBR1 (T-box brain 1 transcription factor) is a negative regulator of FEZF2 and therefore not expressed in the PT neurons, all of which are known to be FEZF2-positive (Chen et al., 2008;Han et al., 2011;McKenna et al., 2011). TBR1 is predominantly expressed in L6 CT neurons and subpopulations of L2/3 and L5 non-PT excitatory neurons instead (Han et al., 2011;McKenna et al., 2011;Matho et al., 2021). To further elucidate the distributions of GFP (Nav1.1)-expressing neurons in neocortex, we performed immunohistochemical staining of TBR1 on Scn1a-GFP mice (Figure 14 and Supplementary file 1ag, ah, ai) and quantitated GFP signal intensities and area sizes of cells ( Figure 15 and Supplementary file 1aj). TBR1-positive cells were predominant in neocortical L6, some in L5 and a few in L2/3. In L5, contrary to the high ratios of GFP-positive cells among FEZF2-positive cells (83% at P15 and 96% at 4W) ( Figure 12B), the ratios of GFP-positive cells among TBR1-positive cells are quite low (11% at P15 and 5% at 4W) ( Figure 14B, middle panels and Supplementary file 1ah). Soma sizes of TBR1/GFP-double positive cells were smaller than those of TBR1-negative/GFPpositive cells (Figure 15, middle panel and Supplementary file 1aj). In L6 where a major population of TBR1-positive neurons locate, the ratios of GFP-positive cells among TBR1-positive cells are still low (15% at P15 and 26% at 4W) ( Figure 14B, middle panels and Supplementary file 1ah). Soma sizes of TBR1/GFP-double positive cells were also smaller than those of TBR1-negative/GFP-positive cells, and TBR1/GFP-double positive cells showed lower intensity of GFP immunosignals compared to The online version of this article includes the following source data for figure 5: Source data 1. Numerical source data for Figure 5F.   file 1aj). We additionally performed triple immunostaining for TBR1, Nav1.1, and ankyrinG on Scn1a-GFP mice (Figure 14figure supplement 1 and Supplementary file 1ak, al, am). Notably, the ratios of Nav1.1-positive cells among TBR1-positive cells are 0% in all layers (Figure 14-figure supplement 1B, right panel and  Supplementary file 1am). These results indicate that the major population of TBR1-positive cells do not express Nav1.1.
Taken together, these results indicate that most TBR1-positive neurons including L6 CT neurons do not express Nav1.1 but expresses Nav1.2.
As a whole, above results showed that a minor subpopulation of L2/3 CC and L5 PT neurons express Nav1.1 while the majority of L2/3 CC, L5/6 CS, and L6 CT neurons express Nav1.2. A breakdown of L5 neuron subtypes is specifically described in Figure 14-figure supplement 4.

Discussion
In our present study, we developed Scn1a promoter-driven GFP mice in which the expression of GFP replicates that of Nav1.1. All PV-INs and most of SST-INs were GFP-positive in the neocortex and hippocampus of the Scn1a-GFP mouse, being consistent with the previous reports of Nav1.1 expression in those inhibitory neurons (Ogiwara et al., 2007;Lorincz and Nusser, 2008;Ogiwara et al., 2013;Li et al., 2014;Tai et al., 2014;Tian et al., 2014;Yamagata et al., 2017). All Nav1.1positive cells were GFP-positive. Reversely all GFP-positive cells were also Nav1.1-positive in the hippocampus, but in the neocortex only a half of GFP-positive cells were Nav1.1-positive. This is largely because in the hippocampus Nav1.1 expression is restricted to inhibitory neurons, but in the neocortex Nav1.1 is expressed not only in inhibitory but also in a subpopulation of excitatory neurons in which Nav1.1 expression is low and not easily detected immunohistochemically by anti-Nav1.1 antibodies. In neocortex, one-third of GFP-positive cells were GABAergic cells such as PV-INs and SST-INs, and the remaining two-third were excitatory neurons. GFP signals were especially intense in PV-positive cells indicating strong Nav1.1 expression in those cells, while GFP signals in SST-positive cells were similar to those in excitatory neurons. In addition, extensive immunostaining analyses using projection neuron markers FEZF2 and TBR1 together with anti-Nav1.2 antibody also revealed that most L5 PT neurons and a minor subpopulation of L2/3 CC neurons express Nav1.1 while the majority of L6 CT, L5/6 CS, and L2/3 CC neurons express Nav1.2.
The above observations should contribute to understanding of neural circuits responsible for diseases such as epilepsy and neurodevelopmental disorders caused by SCN1A and SCN2A mutations. Dravet syndrome is a sporadic intractable epileptic encephalopathy characterized by early onset (6 months to 1 year after birth) epileptic seizures, intellectual disability, autistic features, ataxia, and increased risk of sudden unexpected death in epilepsy (SUDEP). De novo loss-of-function mutations of SCN1A are found in more than 80% of the patients (Claes et al., 2001;Sugawara et al., 2002;Fujiwara et al., 2003;Dravet et al., 2005;Depienne et al., 2009;Meng et al., 2015). In mice, loss-of-function Scn1a mutations caused clinical features reminiscent of Dravet syndrome, including Magnified images outlined in (A) are shown in (B-D) for antisense probes, and (E-G) for sense probes. o, stratum oriens; p, stratum pyramidale; r, stratum radiatum; lm, stratum lacunosum-moleculare; m, stratum moleculare; g, stratum granulosum, CA1, cornu ammonis 1; DG, dentate gyrus. Scale bars: 500 µm (A), 50 µm (B-G).  early-onset epileptic seizures, hyperactivity, learning and memory deficits, reduced sociability and ataxic gaits and premature sudden death (Yu et al., 2006;Ogiwara et al., 2007;Oakley et al., 2009;Cao et al., 2012;Han et al., 2012;Kalume et al., 2013;Ito et al., 2013). As also shown in the present study, Nav1.1 is densely localized at AISs of inhibitory cells such PV-IN (Ogiwara et al., 2007;Ogiwara et al., 2013;Li et al., 2014;Tai et al., 2014) and selective elimination of Nav1.1 in PV-IN in mice leads to epileptic seizures, sudden death, and deficits in social behavior and spatial memory Tatsukawa et al., 2018). It is thus plausible that Nav1.1 haplo-deficiency in PV-IN plays a pivotal role in the pathophysiology of many clinical aspects of Dravet syndrome. Notably, mice with selective Nav1.1 reduction in global inhibitory neurons were at a greater risk of lethal seizure than heterozygote null mice (Nav1.1 KO/+), and the mortality risk of mice with Nav1.1 haplo-deficiency in inhibitory neurons was significantly decreased or improved with additional Nav1.1 reduction in dorsal telencephalic excitatory neurons . These observations indicate beneficial effects of Nav1.1 deficiency in excitatory neurons for epileptic seizures and sudden death. Because of the absence of Nav1.1 in hippocampal excitatory neurons (Ogiwara et al., 2007;Ogiwara et al., 2013;Yamagata et al., 2017) and the present study, the ameliorating effect was most possibly caused by Nav1.1 haploinsufficiency in neocortical excitatory neurons. Kalume et al., 2013 reported that parasympathetic hyperactivity is observed in Nav1.1 haplo-deficient mice and it causes ictal bradycardia and finally result in seizure-associated sudden death.

Figure 6 continued
Our present finding of Nav1.1 expression in L5 PT projection neurons which innervate the vagus nerve may possibly elucidate the ameliorating effects of Nav1.1 haploinsufficiency in neocortical excitatory neurons for sudden death of Nav1.1 haplo-deficient mice and may contribute to the understanding of the neural circuit for SUDEP in patients with Dravet syndrome. Further studies including retrograde tracing and electrophysiological analyses are needed.
We previously proposed that impaired CS excitatory neurotransmission causes epilepsies in Scn2a haplo-deficient mouse (Miyamoto et al., 2019). Our present finding of Nav1.2 expression in CS neurons is consistent and further support the proposal. Because SCN2A has been well established as one of top genes which show de novo loss-of-function mutations in patients with ASD (Hoischen et al., 2014;Johnson et al., 2016) and because impaired striatal function was suggested in multiple ASD animal models (Fuccillo, 2016), our finding of Nav1.2 expression in CS neurons may also contribute to the understanding of neural circuit for ASD caused by SCN2A mutations.
In summary, the present investigations using a newly developed Scn1a promoter-driven GFP mice together with anti-Nav1.1/Nav1.2 antibodies and neocortical neuron markers revealed the cellular expression of Nav1.1 and Nav1.2 in more detail. Further developments of mice containing fluorescent protein reporters driven by promoters for other sodium channel genes and combinatorial analyses of those mice are needed to segregate and redefine their unique functional roles in diverse neuronal populations of complexed neural circuits.

Animal work statement
All animal experimental protocols were approved by the Animal Experiment Committee of Nagoya City University and RIKEN Center for Brain Science. Mice were handled in accordance with the guidelines of the Animal Experiment Committee.
The online version of this article includes the following source data for figure 7: Source data 1. Numerical source data for Figure 7C. org). A GFP reporter cassette, comprising a red-shifted variant GFP cDNA and a downstream polyadenylation signal derived from pIRES2-EGFP (Takara Bio), was inserted in-frame into the initiation codon of the Scn1a coding exon 1 using the Red/ET Recombineering kit (Gene Bridges), according to the manufacturer's instructions. A correctly modified BAC clone verified using PCR and restriction mapping was digested with SacII, purified using CL-4B sepharose (GE Healthcare), and injected into pronuclei of C57BL/6J zygotes. Mice carrying the BAC transgene were identified using PCR with primers: mScn1a_TG_check_F1, 5′-TGTT CTCC ACGT TTCT GGTT -3′, mScn1a_TG_check_R1, 5′-TTAG CCTT CTCT TCTG CAAT G-3′, and EGFP_R1, 5′-GCTC CTGG ACGT AGCC TTC-3′ that detect the wildtype Scn1a allele as an internal control (186 bp) and the inserted transgene (371 bp). Of 15 independent founder lines that were crossed with C57BL/6J mice, 12 lines successfully transmitted the transgene to their progeny. Of 12 founders, two lines (#184 and 233) that display much stronger green fluorescent intensity compared with other lines were selected, and maintained on a congenic C57BL/6J background. The mouse lines had normal growth and development. The line #233 has been deposited to the RIKEN BioResource Research Center (https://web.brc.riken.jp/en/) for distribution under the registration number RBRC10241. Slc32a1-Cre BAC transgenic mice and loxP flanked transcription terminator cassette CAG promotor-driven tdTomato transgenic mice Ai14 (B6. Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J, Stock No: 007914, The Jackson Laboratory, USA) were maintained on a C57BL/6J background. To generate triple mutant mice (Scn1a-GFP, Slc32a1-Cre, Ai14), heterozygous Scn1a-GFP and Slc32a1-Cre mice were mated with homozygous Rosa26-tdTomato transgenic mice.

Fluorescence and immunofluorescence quantification
For quantification of inhibitory neurons in GFP-positive cells, we used Scn1a-GFP/Slc32a1-Cre/Ai14 mice at 4W. We acquired multiple color images of primary motor cortex and hippocampus from three parasagittal sections per animal. Six images per region of interest were manually counted and summarized using Adobe Photoshop Elements 10 and Excel (Microsoft). On immunofluorescence quantification, we used Scn1a-GFP mice at P15 and/or 4W for the quantification of immunosignals. For quantification of GFP-, NeuN-, PV-, SST-, Nav1.1-, Nav1.2-, FEZF2-, or TBR1-positive cells, we acquired multiple color images of primary motor cortex and hippocampus from three parasagittal sections per animal. Six to nine images per region of interest were manually quantified and summarized. For quantification of PV-, SST-, FEZF2-, or TBR1-positive cells, intensity and area size of GFP fluorescent signals were measured by Fiji software. Statistical analyses were performed by one-way analysis of variance followed by Tukey-Kramer post hoc multiple comparison test using Kyplot 6.0 (KyensLab Inc). p value smaller than 0.05 was considered statistically significant. Data are presented as the mean ± standard error of the mean.

In situ hybridization
Frozen sections (30 µm) of PFA-fixed mouse brains at 4W were incubated in 0.3% H 2 O 2 in PBS for 30 min at RT to quench endogenous peroxidases, and mounted on glass slides. The sections on slides hippocampal CA1, CA2/3, and DG are plotted (see also Supplementary file 1n). (B) Box plots represent values for the intensity and area size in each cell type (see also Supplementary file 1o, p). Cross marks indicate mean values in each cell type. Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer post hoc multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001. Note that GFP signal intensities of PV/GFP-double positive cells were significantly higher than that of SST/GFP-double positive cells and PV/SST-negative/GFP-positive cells (all layers), while GFP signal intensities of SST/GFP-double positive cells were similar to PV/SST-negative/GFP-positive cells in neocortex. In hippocampus, GFP signal intensities of SST/GFP-double positive cells were significantly lower than that of SST-negative/GFP-positive cells at CA2/3 and DG. CA1, CA2/3, and DG: cornu ammonis 1, 2 plus 3, dentate gyrus. a.u., arbitrary unit; +, positive; −, negative.
The online version of this article includes the following source data for figure 9: Source data 1. Numerical source data for Figure 9. The online version of this article includes the following source data for figure 10: Source data 1. Numerical source data for Figure 10B.
were UV irradiated with 1250 mJ/cm 2 (Bio-Rad), permeabilized with 0.3% Triton X-100 in PBS for 15 min at RT, and digested with 1 µg/ml proteinase K (Nacalai Tesque) in 10 mM Tris-HCl and 1 mM EDTA, pH 8.0, for 30 min at 37°C, washed twice with 100 mM glycine in PBS for 5 min at RT, fixed with 4% formaldehyde in PBS for 5 min at RT, and acetylated with 0.25% acetic anhydride in 100 mM The online version of this article includes the following source data for figure 11: Source data 1. Numerical source data for Figure 11B. Source data 1. Numerical source data for Figure 12B.        containing the Biotin solution (Vector Laboratories) overnight at 60°C in a humidified chamber. The hybridized sections were washed with 50% formamide in 2× Saline-sodium citrate buffer (SSC) for 15 min at 50°C twice, incubated in TNE (1 mM EDTA, 500 mM NaCl, 10 mM Tris-HCl, pH 8.0) for 10 min at 37°C, treated with 20 µg/ml RNase A (Nacalai Tesque) in TNE for 15 min at 37°C, washed 2× SSC twice for 15 min each at 37°C twice and 0.2× SSC twice for 15 min each at 37°C. After washing twice in a high stringency buffer (10 mM Tris, 10 mM EDTA, and 500 mM NaCl, pH 8.0) for 10 min graphs indicating the percentage of cells with TBR1-and GFP-positive/negative nuclei and somata per GFP-positive cells and/or TBR1-positive nuclei (left panels) (see also Supplementary file 1ag), the percentage of cells with GFP-positive/negative somata per cells with TBR1-positive nuclei (middle panels) (see also Supplementary file 1ah), and the percentage of cells with TBR1-positive/negative nuclei per cells with GFP-positive somata (right panels) (see also Supplementary file 1ai) in L2/3, L5, and L6. Cells in primary motor cortex of Scn1a-GFP mouse at P15 and 4W were counted. Note that 86% (P15) and 95% (4W) of cells with TBR1-positive cells are GFP-negative in L5 (middle panels), and 90% (P15) and 96% (4W) of cells with GFP-positive cells are TBR1-negative in L5 (right panel). L2/3, L5, L6: neocortical layer II/III, V, VI. +, positive; −, negative.
The online version of this article includes the following source data and figure supplement(s) for figure 14: Source data 1. Numerical source data for Figure 14B.        . TBR1-positive cells have lower green fluorescent protein (GFP) signal intensities in Scn1a-GFP mouse neocortex. Scatter plots of intensities and area sizes of GFP immunosignals in GFP-positive cells with TBR1-positive/negative nuclei. Cells at primary motor cortex in parasagittal sections from 4W Scn1a-GFP mouse (line #233) were analyzed. TBR1-positive (orange squares) and negative (black circles) cells in neocortical L2/3, L5, and L6 are plotted (see also Supplementary file 1aj). Note that GFP signal intensities of TBR1/GFP-double positive cells were significantly lower than that of TBR1-negative/GFP-positive cells (L6), and signal area size of TBR1/GFP-double positive cells was significantly smaller than that of TBR1-negative/GFPpositive cells (L5, L6). Statistical significance was assessed using t-test. a.u., arbitrary unit; +, positive; −, negative.
The online version of this article includes the following source data for figure 15: Source data 1. Numerical source data for Figure 15. each at RT, the sections were blocked with a blocking buffer [20 mM Tris and 150 mM NaCl, pH 7.5 containing 0.05% Tween-20, 4% BlockAce (DS Pharma Biomedical) and 0.5× Blocking reagent (Roche Diagnostics)] for 1 hr at RT, and incubated with the alkaline phosphatase-conjugated sheep anti-DIG (1:500; 11093274910, Roche Diagnostics) and biotinylated rabbit anti-DNP (1:100; BA-0603, Vector Laboratories) antibodies in a blocking buffer overnight at 4 °C, followed by incubation with the biotinylated goat anti-rabbit antibody (1:200; BA-1000, Vector Laboratories) in a blocking buffer at RT for 1-2 hr. The probes were visualized using the NBT/BCIP kit (Roche Diagnostics), VECTASTAIN Elite ABC kit (Vector Laboratories), and ImmPACT DAB substrate (Vector Laboratories).
The DIG-labeled RNA probes for Scn1a designed to target the 3′-untranlated region (nucleotides 6488-7102 from accession number NM_001313997.1) were described previously (Ogiwara et al., 2007), and synthesized using the MEGAscript transcription kits (Thermo Fisher Scientific) with DIG-11-UTP (Roche Diagnostics). The DNP-labeled RNA probes for GFP were derived from the fragment corresponding to nucleotides 1256-1983 in pIRES2-EGFP (Takara Bio), and prepared using the MEGAscript transcription kits (Thermo Fisher Scientific) with DNP-11-UTP (PerkinElmer). The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. Additional files